Carbohydrates are an important class of biological compounds. The term "saccharides" encompasses a wide variety of carbohydrate-containing compounds. These include polysaccharides, oligosaccharides, glycoproteins and glycosides with non-carbohydrate aglycones. Biological macromolecules composed of protein or lipids containing oligosaccharide moieties are collectively known as glycoconjugates. The carbohydrate moiety provides many biological functions.
In cells, carbohydrates function as structural components where they regulate viscosity, store energy, or are key components of the cell surface. The complex oligosaccharide chains of various glycoconjugates (especially glycoproteins and glycolipids) mediate or modulate a variety of biological processes. For a general review of the bioactivity of carbohydrates see: (a) Biology of Carbohydrates, Volume 2, Ginsburg et al, Wiley, N.Y. (1984); and (b) P. W. Macher et al, Annual Review of Biochemistry, Volume 57, page 785, 1988). Among other things, it is known that:
(a) carbohydrate structures are important to the stability, activity, localization and degradation of glycoproteins; PA1 (b) certain oligosaccharide structures activate plant secretion of antimicrobial substances; PA1 (c) glycoconjugates are frequently found on the surfaces of various cells and are important, inter alia, to cell interactions with the surroundings since they function as receptors or regulators when bonded to cell surfaces of, for example, peptides, hormones, toxins, viruses, bacteria and during cell-cell interaction; PA1 (d) carbohydrate structures are antigenic determinants (for example, blood group antigens); PA1 (e) carbohydrates function as cell differentiating antigens during normal tissue development; PA1 (f) carbohydrates are important in oncogenesis since specific oligosaccharides have been found to be cancer-associated antigenic determinants; and PA1 (g) oligosaccharides are important to sperm/egg interaction and to fertilization. PA1 (a) the development of novel diagnostics and blood typing reagents; PA1 (b) the development of a novel type of therapy as an alternative to antibiotics, based on the prevention of the adhesion of bacteria and viruses to cell surfaces by means of specific oligosaccharides; and PA1 (c) the use of oligosaccharides to stimulate plant growth and provide protection against certain plant pathogens. PA1 (a) Host, host cell or host animal: These terms are used to refer to the cell or mammal which is responsible for the biosynthesis of biological material. PA1 (b) Homologous: This word means that the entity thus characterized is normally present or produced by the host. PA1 (c) Heterologous: This word means that the entity thus characterized is not normally present or produced by the host. In other words, the entity thus characterized is foreign to the host. PA1 (d) Catalytic activity: This term is used to refer to the inherent property of certain biological compounds to facilitate chemical change in other substances. PA1 (e) Catalytic entity: This term is used to refer to biological compounds which inherently possess catalytic activity which results in the production of new, different or altered compounds. Examples hereof are enzymes and antibodies. An enzyme is a biochemical catalyst of a specific biochemical reaction. An enzyme product is formed as a result of the enzyme's catalytic activity on a substrate material. PA1 (f) Genome: This word is used to refer to the complete genetic material found in the host. This material is arranged in chromosomes. PA1 (g) Gene: This word refers to a functional portion of the genome which is responsible for the biosynthesis of a specific biological entity. PA1 (h) Insertion: This word is used to refer to the process whereby a portion of heterologous DNA or a heterologous gene that is introduced into the genome of a host. The DNA which is inserted is referred to as an "insert". PA1 (i) Transgene: This refers to heterologous genetic material which is transferred by insertion from the genome of one animal species to the genome of another animal species. More simply, a transgene is a gene which is heterologous to the host. The transgene encodes a specific biological material. PA1 (j) Transgenic mammal or transgenic host: These terms are used to refer to a mammal or cell which has had a transgene inserted into its genome. As a result of this insertion, the transgenic host produces heterologous biological material that it would not normally synthesize. Heterologous entities are present or are produced by a transgenic host as a result of the insertion of foreign genetic material into the host cell genome. PA1 (k) Primary gene product: This refers to a biological entity which is formed directly as a result of the transcription and translation of a homologous or heterologous gene. Examples thereof include proteins, antibodies, enzymes and the like. PA1 (l) Secondary gene product: This refers to a product which is formed as a result of the biological activity of a primary gene product. An example thereof, is an oligosaccharide which is formed as a result of the catalytic activity of an enzyme. PA1 (m) Biological products: This term is used to refer to products produced or synthesized by a transgenic host as a result of the insertion of a transgene into the genome of the mammal. More specifically, the term means biological products which are secondary gene products. One example hereof, as described below, is human oligosaccharides produced by transgenic cows. Human oligosaccharides are produced as a result of the catalytic activity of human glycosyltransferases. As discovered herein, when the gene encoding human glycosyltransferases is inserted into the murine genome, the resultant transgenic mouse produces a heterologous human glycosyltransferase as the primary gene product. The human glycosyltransferase, using homologous substrate materials, produces oligosaccharides and glycosylated proteins. The oligosaccharide, formed as a result of enzyme activity of the primary gene product, is also properly called a secondary gene product. Glycoconjugates are another example of the class of compounds referred to herein and in the claims referred as "biological products". PA1 (n) Product: This word is used to refer to the secondary gene products of the instant invention and is used as an alternative to "biological product". PA1 (o) Humanized milk: This refers to milk obtained from a non-human mammal which, through alteration of the host genome, is made to produce milk which more closely resembles human milk. One example of humanized milk is cow's milk containing products found in human milk but not normally found in the cow's milk. Human oligosaccharides are produced in cow's milk as a result of the insertion of the gene encoding human glycosyltransferases into the bovine genome. Humanized milk also contains proteins glycosylated with human oligosaccharides. PA1 1) U.S. Pat. No. 5,032,519 to Paulson teaches a method for genetically engineering cells so that they produce soluble and secretable Golgi processing enzymes instead of the naturally occuring membrane-bound enzymes. PA1 2) U.S. Pat. No. 5,047,335 to Paulson teaches the alteration by genetic engineering of the genome of Chinese Hamster Ovary Cells (CHO) so that the CHO cells produce a sialytransferase. PA1 3) International Patent Application No. PCT/US91/08216 teaches a transgene capable of producing hetrologous recombinant proteins in the milk of transgenic bovine species. This published patent application teaches a method for obtaining the primary gene product only. This published patent application also discloses methods of producing and using the altered milk obtained from these transgenic animals. PA1 4) International Patent Application No. PCT/US91/05917 teaches methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments. This published patent application teaches a method for obtaining the primary gene product only. PA1 5) International Patent Application No. PCT/GB87/00458 teaches methods of producing a peptide, said method involving incorporating a DNA sequence coding for the peptide into the gene of a mammal coding for a milk whey protein in such a way that that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application teaches a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal, and also discloses methods of producing and using the altered milk obtained from these transgenic animals. PA1 6) International Patent Application No. PCT/GB89/01343 teaches methods for producing proteinaceous materials in transgenic animals that have genetic constructs integrated into their genomes. The construct comprises a 5'-flanking sequence from a mammalian milk protein gene and DNA coding for a heterologous protein other than a milk protein. This published patent application teaches a method for obtaining only the primary gene product, the heterologous protein, in the milk of the transgenic mammal. PA1 7) European Patent Application No. 88301112.4 teaches methods for targetting specific genes to the mammary gland which results in the efficient synthesis and secretion of biologically important molecules into the milk of these transgenic animals. This published patent application also teaches methods of producing and using the altered milk obtained from these transgenic animals, and a method for obtaining only the primary gene product in the milk of the transgenic mammal. PA1 8) International Patent Application No. PCT/DK93/00024 teaches methods for producing human kappa-casein in the milk of transgenic animals. The genetic construct comprises a 5'-flanking sequence from a mammalian milk protein gene, such as casein or whey acid protein, and DNA coding for human kappa casein. The DNA sequence contains at least one intron. This published patent application teaches a method for obtaining only the primary gene product, the heterologous human kappa casein, in the milk of the transgenic mammal. PA1 9) International Patent Application No. PCT/US87/02069 teaches a method for producing mammals capable of expressing recombinant proteins in their milk. PA1 (a) inserting into the genome of a non-human mammal a heterologous gene encoding the production of a human catalytic entity wherein said catalytic, entity produces a secondary gene product in the milk of said non-human mammal; and PA1 (b) milking said non-human mammal. PA1 (a) inserting into the genome of a non-human mammal a heterologous gene encoding the production of a heterologous catalytic entity wherein said catalytic entity produces a secondary gene product in the milk of said non-human mammal; and PA1 (b) milking said non-human mammal; and PA1 (c) isolating the biological product from said milk. PA1 (a) preparing a transgene, said transgene consisting of at least one expression regulation DNA sequence functional in the mammary secretory cells of said transgenic species, a secretory DNA sequence functional in the mammary secretory cells of said transgenic species and a recombinant DNA sequence encoding a recombinant heterologous catalytic entity, said secretory DNA sequence being operably linked to said recombinant DNA sequence to form a secretory-recombinant DNA sequence and said at least one expression regulation sequence being operably linked to said secretory-recombinant DNA sequence, wherein said transgene is capable of directing the expression of said secretory-recombinant DNA sequence in mammary secretory cells of said transgenic species containing said transgene to produce a recombinant heterologous catalytic entity which when expressed by said mammary secretory cells catalyses the production of secondary gene products in the milk of said transgenic species; PA1 (b) introducing said transgene into the embryonic target cell; transplanting the transgenic embryonal target cell formed thereby or the embryo formed herefrom into a recipient female parent; and PA1 (c) identifying at least one female offspring which is capable of producing said secondary gene products in the milk of said offspring. PA1 (a) preparing a transgene capable of conferring said phenotype when incorporated into the cells of said transgenic non-human mammal PA1 (b) methylating said transgene; PA1 (c) introducing said methylated transgene into fertilized oocytes of said non-human mammal to permit integration of said transgene into the genomic DNA of said fertilized oocytes; PA1 (d) culturing the individual oocytes formed hereby to pre-implantation embryos thereby replicating the genome of each of said fertilized oocytes; PA1 (e) removing at least one cell from each of said preimplantation embryos and lysing said at least one cell to release DNA contained therein; PA1 (f) contacting said released DNA with a restriction endonuclease capable of cleaving said methylated transgene but incapable of cleaving the unmethylated form of said transgene formed after integration into and replication of said genomic DNA; and PA1 (g) detecting which of said cells from said preimplantation embryos contain a transgene which is resistant to cleavage by said restriction endonuclease as an indication of which pre-implantation embryos have integrated said transgene. PA1 (g) cloning at least one of said second hemiembryos; and PA1 (h) to form a multiplicity of transgenic embryos. PA1 (a) A portion encoding the human glycosyltransferase. This portion of the transgene is hereinafter referred to as the "recombinant portion" or "recombinant sequence"; PA1 (b) A signal portion; and PA1 (c) An expression regulation portion.
Isolated oligosaccharides are known to inhibit the agglutination of uropathogenic coliform bacteria with erythrocytes. Other oligosaccharides have been shown to possess potent antithrombic activity by increasing the levels of plasminogen activator. This same biological activity has been used, by covalently attaching these oligosaccharides to the surface of medical instruments, to produce surfaces which have anticoagulation effects. These surfaces are useful in the collection, processing, storage and use of blood. Still other oligosaccharides have found utility as gram positive antibiotics and disinfectants. Further, certain free oligosaccharides have been used in the diagnosis and identification of specific bacteria. A considerable future market is envisaged for fine chemicals based on biologically active carbohydrates.
Universities and industry are at present working intensely on developing the additional uses of biologically active oligosaccharides. These efforts include, but are not limited to:
A large number of oligosaccharide structures have been identified and characterized. The smallest building block or unit of an oligosaccharide is a monosaccharide. The major monosaccharides found in mammalian glycoconjugates are: D-glucose (Glc), D-galactose (Gal), D-mannose (Man), L-fucose (Fuc), N-acetyl-D-galactose amine (GalNAc), N-acetyl-D-glucose amine (GIcNAc) and N-acetyl-D-neuraminic acid (NeuAc). The abbreviations in parentheses are the standard abridged terminology for monosaccharides according to the recommendations of the International Union of Physics, Chemistry and Biology Council; Journal Biological Chemistry, Volume 257, pages 3347-3354, (1982). These abbreviations will be used hereinafter. Despite the relatively small number of fundamental building blocks, the number of possible combinations is very great because both the anomeric configuration (alpha- or .beta.-glycosidic linkage) as well as the position of the O-glycosidic bond can be varied.
Thus, a large variety of oligosaccharide structures can exist. The bioactivity of oligosaccharides is known to be specific in terms of both sugar conformation and composition. Individual monosaccharides provide one element of bioactivity but they also contribute to the overall conformation of the oligosaccharide thereby providing another level of specificity and bioactivity. It is the diversity of glycoconjugates and oligosaccharides that produces biological specificity of certain oligosaccharide structures. However, this diversity also causes a particular problem for the practical utility of these compounds. Glycoconjugates are typically potent immunogens and the biospecificity, as noted above, is determined not only by the particular monosaccharide sequence but also by the nature of the glycosidic bond. Consequently it is often not possible to use oligosaccharide structures found in one animal species in another species. Similar restrictions on use may also apply on an individual basis. For example, since certain blood group antigens are known to be formed from specific oligosaccharides, it is necessary to be especially careful when conjugating a blood group oligosaccharide to a protein and then using that glycoprotein therapeutically. Careful consideration of the potential immunogenicity concerns must be made.
Despite these potential difficulties, it is well accepted that there is a need to produce large quantities of human oligosaccharides and/or glycoconjugates bearing those oligosaccharides. Numerous methods have been contemplated as suitable means for achieving this goal. Such methods include synthesis of oligosaccharides by conventional organic chemistry or the use of enzymes in vitro. Immobilized enzymes are presently the preferred mode for large scale in vitro oligosaccharide production. This is because of an enzyme's high regio- and stereoselectivity, as well as a high catalytic efficiency under mild reaction conditions. The literature discloses a number of enzyme-catalyzed oligosaccharide syntheses. For example, see the scientific review articles by Y. Ichikawa et al, "Enzyme-catalyzed Oligosaccharide Synthesis" in Analytical Biochemistry, Volume 202, pages 215-238, (1992); and K. G. I. Nillson, "Enzymatic Synthesis of Oligosaccharides" Trends in Biotechnology, Volume 6, pages 256-264, (1988). Both hydrolases and transferases have been used to faciltate production of oligosaccharides. The glycosidase enzymes, a subclass of the hydrolases, are especially useful in the synthesis of oligosaccharides by a process of reversing the degradative cycle. In general, however, enzymatic oligosaccharide synthesis is based on the biosynthetic pathway. While the biosynthetic pathway of oligosaccharide synthesis is principally regulated by the gene encoding the production of each glycosyltransferase, the actual oligosaccharide structures are determined by the substrate and acceptor specificity of the individual glycosyltransferases. Oligosaccharides are synthesized by transferring monosaccharides from sugar nucleotide donors to acceptor molecules. These acceptor molecules may be other free oligosaccharides, monosaccharides, or oligosaccharides bound to proteins or lipids.
Enzymatic oligosaccharide synthesis has generally been conducted only on a small scale because the enzymes, particularly the glycosyltransferases from natural sources, have been difficult to isolate. Also, the sugar nucleotide donors are very difficult to obtain from natural sources and are very expensive when derived from organic chemistry synthesis. More recently however, a recycling and reutilization strategy has been developed for synthesizing large quantities of oligosaccharides. U.S. Pat. No. 5,180,674, incorporated herein by reference, discloses a novel affinity chromatography method in which the reaction products are repetitively recycled over the matrix or resin bound glycosyltransferases. Furthermore, recent progress in gene cloning techniques have made several glycosyltransferases available in sufficient quality and quantity to make enzymatic synthesis of oligosaccharides more practical.
The literature is replete with descriptions of recombinant or transgenic expression of a heterologous glycosyltransferase. However, before continuing a discussion of the literature, it is necessary to clarify the meaning of various terms as used herein and in the claims:
As noted above, there is a considerable body of literature which describes the recombinant or transgenic expression of heterologous glycosyltransferases. However, the literature does not disclose or in any other manner suggest production of secondary gene products in the milk of non-human transgenic mammals as claimed in the instant invention. Examples of the literature are:
These publications each teach, in one manner or another, a means for obtaining the primary gene product of the transgene, that gene product being the active protein or enzyme which is encoded by the transgene. This literature discloses transgenic means for obtaining glycosyltransferases in non-human milk. However, none of the aforementioned publications teaches or suggests the use of transgenic animals as a means of obtaining a desired secondary gene product which is the product of the active enzyme. More particularly, however, none of the aforementioned publications teaches or suggests or in any other manner discloses the use of transgenic human glycosyltransferases in non-human milk to produce human oligosaccharides or glycoconjugates bearing those oligosaccharides. These oligosaccharides, which are the product of active glycosyltransferases, are hereinafter referred to as the "secondary gene product". Thus, the various oligosaccharides found in human milk are formed as a direct result of the genetically regulated expression of certain specific glycosyltransferases. In this regard, oligosaccharides may be properly considered to be "secondary gene products" since they are synthesized as a result of the biochemical activity of the primary gene product, the heterologous glycosyltransferase enzymes.
Human milk contains a variety of oligosaccharides and proteins. Free, soluble oligosaccharides are not normally produced by animal cells and tissues with the exception of the highly differentiated lactating mammary glands. Oligosaccharides constitute the major portion of the total carbohydrate content of human and bovine milk. The major carbohydrate constituent of mammalian milk is the disaccharide lactose. Lactose is typically found at a concentration greater than 10 mg/ml and is synthesized by the attachment of galactose to glucose. This reaction is catalyzed by the enzyme, .beta.-1,4 galactosyltransferase. The milk of most mammals, including cows, contains only very small quantities of a few additional oligosaccharides. In contrast, human milk contains substantial amounts of a number of additional soluble oligosaccharides that are larger than lactose. All human oligosaccharides are synthesized by the sequential addition of monosaccharides to lactose. Representative oligosaccharides found in human milk are shown in Table 1.
TABLE 1 __________________________________________________________________________ OLIGOSACCHARIDES PRESENT IN HUMAN MILK Structure Common Name Concentration (mg/liter) __________________________________________________________________________ Gal.beta.-1,4-Glc Lactose 50,000 Fuc-a-1,2-Gal-.beta.-1,4-Glc 2-fucosyllactose 200 Gal-.beta.-1,3-GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc Lacto-N-tetraose 400 Gal-.beta.-1,4-GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc Lacto-N-neotetraose 60 Fuc-a-1,2-Gal-.intg.-1,3-GlcNAc-.beta.- Lacto-N-fucopentaose I 200 1,3-Gal-.beta.-1,4-Glc Gal-.beta.-1,3Fuc-a-1,4!GlcNAc-.beta.- Lacto-N-fucopentaose II 20 1,3-Gal-.beta.-1,4-Glc Gal-.beta.-1,4Fuc-a-1,3GlcNAc-.beta.- Lacto-N-fucopentoase III 50 1,3-Gla-.beta.-1,4-Glc Fuc-a-1,2-Gal-.beta.-1,3Fuc-a-1,4!- Lacto-N-difucopentaose I 25 GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc NeuAc-a-2,6-Gal-a-1,4-Glc 6-sialyllactose 25 10. NeuAc-a-2,3-Gal-.beta.-1,4-Glc 3-sialyllactose 10 NeuAc-a-2,3-Gal-.beta.-1,3-R Sialyltetrasaccharide a 10 Gal-.beta.-1,3NeuAc-a-2,6!GlcNAc- Sialyltetrasaccharide b 35 .beta.-1,3-R NeuAc-a-2,6-Gal-.beta.-1,4-GlcNAc- Sialyltetrasaccharide c 50 .beta.-1,3-R NeuAc-a-2,3-Gal-.beta.-1,3NeuAc-a-2,6!- Disialyltetrasaccharide 60 GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc NeuAc-a-2,3-Gal-.beta.-1,3Fuc-a-1,4!- Sialyl Lacto-N-fucopentaose 50 GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc __________________________________________________________________________ -a-: denotes an alpha glycosidic linkage R: Gal1,4-Glc
The oligosaccharides in human milk are present as a result of the activity of certain specific glycosyltransferases found in human mammary tissue. For example, the alpha 1,2 linked fucose residues in structures 2,5, and 8 are produced by a unique human fucosyltransferase and characterize a phenotype known in the field of immunohematology as "secretors". These individuals are thus characterized because they synthesize human blood group substances in their salivary and other mucus secretions where the oligosaccharides are covalently attached to various proteins.
The alpha 1,4 linked fucose residues in structures 6,8, and 15 are formed as a result of the enzymatic action of a different fucosyltransferase. These oligosaccharides represent a phenotype present in individuals characterized as having a "Lewis positive" blood type. Such individuals use this fucosyltransferase to synthesize an oligosaccharide structure which corresponds to a human blood group antigen. This oligosaccharide is also found in the saliva, and other mucus secretions, and covalently attached to lipids found on the membrane of red blood cells of "Lewis positive" individuals. Structure 5 is related to the H-antigen of the ABO blood group; structure 6 is the "Lewis a" blood group antigen; structure 8 is the "Lewis b" blood group antigen.
At least fifteen human milk proteins have been identified. Some of these proteins are generally recognized to be glycosylated, i.e. they are covalently attached to certain specific oligosaccharides. The particular oligosaccharides which are covalently attached to the protein are the same as, or similar to, the oligosaccharides described above, and their formation is the result of normal genetically regulated expression of certain specific glycosyltransferase genes. The presence of a heterologous glycosyltransferase would also affect the post-translational modification of proteins. The proteins glycosylated by a heterologous glycosyltransferase are also properly known as "secondary gene products". Both homologous as well as heterologous proteins would be modified by the glycosyltransferase in a manner different from that resulting from the activity of homologous glycosyltransferases.
It has long been known that these oligosaccharides and glycosylated proteins, promote the growth of desirable bacteria in the human intestinal tract. It is also believed that the oligosaccharides in human milk inhibit the attachment of harmful microorganisms to the mouth and throat. These human oligosaccharides and specifically glycosylated proteins are absent from, or present in markedly different amounts in, bovine milk. Further, as noted previously, bovine milk contains predominantly lactose only. Human milk contains not only lactose but also numerous other oligosaccharides. Also, the amino acid composition of human milk proteins is significantly different from the amino acid composition of the corresponding cow's milk proteins. As a consequence, infants fed infant formula which comprises cow's milk may be more susceptible to intestinal disturbances such as diarrhea, or their blood plasma amino acid ratios and levels may differ from breast fed infants. For the same reasons, elderly, immunocompromised and critically ill patients also have an urgent need for the availability of a nutritional product which biochemically closely resembles the composition of human milk.
The complicated chemistry of human milk proteins and oligosaccharides has made their large scale synthesis extremely difficult. Before they can be incorporated into commercial nutritional product, a practical method for obtaining large amounts of glycosylated human milk proteins and oligosaccharides must be devised. One potential solution to this problem is the use of transgenic animals, more particularly transgenic cows which express genes or cDNAs encoding enzymes which catalyse the formation of oligosaccharides and/or proteins glycosylated with the same human oligosaccharides. Transgenic milk-bearing domestic animals, such as rabbits, pigs, sheep, goats and cows, are herein proposed as a means of producing milk containing human oligosaccharides and proteins glycosylated with human oligosaccharides. More particularly, transgenic cows are highly suitable for the production of oligosaccharides and recombinant proteins, because a single cow can produce more than 10,000 liters of milk containing as much as 300 kilograms of protein (mainly casein) per year at a very minimal cost. Thus, transgenic cows appear to be a less costly production route than other recombinant protein production methods since investment in fermentation facilities would not be required. Also, cow mammary glands are more cost effective than cultured cells, are likely to produce continuously and since milk is collected several times a day, the time between the actual synthesis and harvest can be as short as a few hours. The cow's genetic stability is greater than microbial or cell based production systems. Also, cows are relatively easy to reproduce using artificial insemination, embryo transfer and embryo cloning techniques. Further, downstream processing of cow's milk containing human transgenic proteins may require little or no purification. Publications teaching such methods are referred to below. However, none of these publications teaches, discloses or in any other manner suggests production of secondary gene products in the milk of non-human transgenic mammals as claimed in the instant invention.
"Molecular Farming: Transgenic Animals as Bioreactors" by J. Van Brunt, Biotechnology, Volume 6, page 1149-1154, 1988, describes the alteration of the genome of various large domestic milk bearing animals yielding transgenic animals capable of producing various heterologous entities. This publication suggests methods for obtaining the primary gene product only.
International Patent Application No. PCT/US91/08216 describes a transgene capable of producing heterologous recombinant proteins in the milk of transgenic bovine species. This published patent application teaches a method for obtaining the primary gene product only. This application also discloses methods of producing and using the altered milk obtained from these transgenic animals.
International Patent Application No. PCT/GB87/00458 describes methods for producing a peptide, said method involving incorporating a DNA sequence coding for the peptide into the gene of a mammal coding for a milk whey protein in such a way that that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application teaches a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal. This application also-discloses methods of producing and using the altered milk obtained from these transgenic animals.
International Patent Application No. PCT/GB89/01343 teaches methods of producing proteinaceous materials in transgenic animals that have genetic constructs integrated into their genome. The construct comprises a 5'-flanking sequence from a mammalian milk protein gene and DNA coding for a heterologous protein other than a milk protein. This published patent application teaches a method for obtaining only the primary gene product, the heterologous protein, in the milk of the transgenic mammal.
European Patent Application No. 88301112.4 describes methods for targeting specific genes to the mammary glands which results in the efficient synthesis and secretion of biologically important molecules into the milk of these transgenic animals. This published application also discloses methods of producing and using the altered milk obtained from these transgenic animals and teaches a method for obtaining only the primary gene product in the milk of the transgenic mammal.
International Patent Application No. PCT/US87/02069 teaches a method for producing mammals capable of expressing recombinant proteins in the milk of lactating animals. This patent application does not disclose or in any other manner suggest production of secondary gene products in the milk of non-human transgenic mammals as claimed in the instant invention.
While transgenic animals can be used for the production of large quantities of human proteins, they have not been used for the production of secondary gene products, such as human oligosaccharides or proteins and lipids glycosylated with certain specific oligosaccharides, or human milk proteins and lipids glycosylated with certain specific oligosaccharides. None of the aforementioned publications discloses or suggests a method for producing human oligosaccharides and glycoconjugates in non-human mammalian milk. The aforementioned publications also do not disclose or suggest a method for obtaining glycoconjugates in non-human mammalian milk wherein the glycosylation is with the desired oligosaccharides. Achieving this result requires that the genome of non-human milk-bearing mammals be altered so as to ensure that the mammary tissue which selectively expresses a desired human glycosyltransferase which would then glycosylate certain proteins with the desired oligosaccharide. This approach requires the DNA encoding the desired human glycosyltransferase be incorporated into said genome. The literature also does not disclose or suggest a method for obtaining glycosylated human proteins in non-human mammalian milk wherein the glycosylation is with the desired oligosaccharides. The literature also does not disclose or suggest a method for obtaining glycosylated human milk proteins in non-human mammalian milk wherein the glycosylation is with the desired oligosaccharide moieties. Achieving this result would require that the genome of non-human milk-bearing mammals to be altered so as to ensure that its mammary tissue selectively expresses both the human glycosyltransferase as well as the desired human proteins which are then appropriately glycosylated with the desired oligosaccharides by the active human glycosyltransferase. This approach requires not only that the DNA encoding the desired glycosyltransferase be inserted into said genome but also that the DNA encoding the desired human proteins also be incorporated into said genome.
Accordingly, it is an aspect of the present invention to provide methods for detecting succesful transgenesis of fertilized oocytes prior to implantation, such that the transplanted oocytes contain the genetic constructs required to achieve the desired glycosylation and oligosaccharide production.
It is also an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing human glycosyltransferases that are secreted extracellularly by the mammary tissue of said mammalian species.
Further, it is also an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing human glycosyltransferases that are secreted extracellularly by the mammary tissue into the milk produced by said mammalian species.
In addition, it is an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing glycosylated human proteins and oligosaccharides that are secreted extracellularly by the mammary tissue into the milk produced by said mammalian species.
The present invention also relates to transgenic non-human milk bearing mammalian species which are capable of producing glycosylated human milk proteins and lipids in the milk of such transgenic animals.
It is also an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing human oligosaccharides in the milk of such transgenic animals.
The present invention also relates to food formulations containing glycosylated human proteins, lipids and oligosaccharides from such transgenic milk.
The present invention also relates to pharmaceutical, medical diagnostic and agricultural formulations containing glycosylated proteins, lipids and oligosaccharides obtained from the milk of transgenic animals.
It is also an aspect of the present invention to provide transgenic bovine species that are capable of producing glycosylated proteins such as glycosylated human milk proteins and lipids in their mammary glands.
It is a further aspect of the present invention to provide transgenic bovine species that are capable of producing human oligosaccharides in the milk of such transgenic cows.
The present invention also relates to food formulations containing glycosylated proteins, lipids and oligosaccharides from such transgenic bovine milk.
The present invention also relates to pharmaceutical, medical diagnostic and agricultural formulations containing glycosylated proteins, lipids and oligosaccharides obtained from the milk of transgenic cows.